In this work, a convenient ratiometric fluorescent platform was designed to measure organophosphorus pesticides (OPs) based on acetylcholinesterase (AChE), acetylthiocholine (ATCh), manganese dioxide nanosheets (MnO2), near-infrared carbon dots (RCDs) and o-phenylenediamine (OPD). In this platform, a direct oxidation of OPD by MnO2 generated the luminescent product 2,3-diaminophenolazine (DAP) through intrinsic oxidase activity, while RCDs served as a fluorescent reference indicator. In the presence of AChE and ATCh, the enzymatic hydrolysate thiocholine (TCh) would reduce MnO2 nanosheets to Mn2+, leading to the quenching of DAP fluorescence. On the other hand, OPs can inhibit the catabolism of ATCh by AChE thus acting as a recognizer of OPs. According to these reactions, OPs were quantitatively analyzed by the intensity ratio of fluorescence emitted from RCDs and DAP (F560/F676). The constructed platform can detect OPs with the range of 0.2–0.6 μM with a detection limit of 4.3 nM. Figure A ratiometric fluorescent probe based on carbon dots was obtained and using it to determine the concentration of organophosphorus pesticides